CN115803083A - Novel lipase - Google Patents

Abstract

The present invention relates to a lipase comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO 1, or a functional fragment thereof encompassing position Q55, with the proviso that said lipase has a substitution at position Q55 of an amino acid with a basic side chain at neutral pH (fig. 1).

Description

Novel lipase

Technical Field

The present application relates to novel lipases.

Background

Lipid digestion defects and disorders play an increasingly important role in general medical and medical practice. In many cases, such digestive disorders are the result of more or less pronounced defects of the so-called pancreatin. In a healthy state, these enzymes are synthesized in the pancreas by highly differentiated cells, so-called acinar cells, and secreted into the duodenum by extracellular secretion via the fluid glands and the main pancreatic ducts. The total daily amount of pancreatic juice secretion is about 2 liters. In addition to fat-digesting lipases, pancreatic secretion also comprises enzymes for digesting proteins (trypsin, chymotrypsin and carboxypeptidase) and carbohydrates (α -amylase). The secretion of pancreatic enzymes is precisely controlled by endogenous control mechanisms by hormonal means such as gastrin, secretin and secretin. This control system can be disturbed by a number of factors, resulting in a decrease in pancreatic enzyme secretion and a complete resolution of pancreatic exocrine function. This in turn results in chyme not being digested in the small intestine and digestive disorders occurring. This disease of the digestive tract, also known as Exocrine Pancreatic Insufficiency (EPI), may have different causes. In addition to drug-induced dyspepsia, chronic atrophic gastritis and chronic pancreatitis, often caused by alcohol consumption, surgically-induced disorders (e.g., perono I and II, vagotomy, pancreatectomy), and cystic fibrosis are the causes of pancreatic insufficiency. In any case, chronic digestive disorders have considerable socio-medical and, hence, economic significance, since symptoms often result in patients with uncharacteristic and shortened life expectancy.

Pancreatic digestive disorders, and in particular EPI, cause a great deal of discomfort to the patient, such as diarrhea, stool mass, satiety, epigastric discomfort, weight loss, and the like.

Regardless of the etiology and characterization of pancreatic dyspepsia or EPI, it is critical that replacement therapy with enzymes be initiated as soon as EPI is diagnosed in order to avoid morbidity and mortality associated with malnutrition. This means that the deficient enzymes (mainly lipases, proteases and amylases) have to be provided externally. In therapy, the enzymes are taken orally by the patient, mainly at the time of eating and through the stomach and to the small intestine, where they carry out digestion of chyme and thus assume the function of the endogenous pancreatic enzymes that are lacking.

For the treatment of digestive disorders based on pancreatin deficiency, pancreatin replacement therapy (PERT) is generally used, which is based on the replacement/substitution of the main enzymes, i.e. lipases and proteases. For PERT, various enzyme preparations have been marketed. These are based in part on pancreatin from pigs, e.g. preparations

Preparations containing pancreatin, known as pancreatin products or PEPs, are obtained in large part from the pancreas of slaughtered pigs, for example pigs. The final product of the preparation process is pancreatin. PEPs are composed of porcine lipase, amylase, and protease and are used in patients with EPI secondary to cystic fibrosis, chronic pancreatitis, and pancreatectomy.

The pig origin PEP cannot be used for digestive disorder patients with pig protein allergy. In addition, pigs are regarded as a natural reservoir for human pathogenic influenza viruses and a large number of viruses of porcine origin, so that contamination of pancreatic juice with such viruses cannot be ruled out. In other words, pancreatic tissue may present slaughter waste, which, if not further processed, may show high viral contamination. Thus, pancreatic tissue, pancreatin and PEP can also be contaminated with porcine-derived viruses based on their natural origin. It must be stressed that the technical requirements of human medicine require the international coordination council (ICH) to set extremely high standards in its guideline ICH topoc Q5A (R1) and to most reasonably ensure that the product is free from viral contamination. The drug evaluation and research Center (CDER) of the FDA in the united states has mandated that each must be required for a lipase-containing PEP to enforce risk mitigation strategies.

This is due to the risk of PEP contamination with porcine parvovirus and porcine circovirus as well as a large number of porcine viruses known to be human pathogens.

For these reasons, there is a need for better-defined, less-risk lipases for use in pancreatic enzyme replacement therapy.

Another problem is that each preparation used must contain a sufficient amount of enzyme. In addition, the enzymes must be provided in an enteral formulation, have a small particle size and be fully bioavailable within the digestive tract.

In practice, the daily dose for a patient can become very large. The starting dose is about 50,000-75,000 units lipase at meal time and about 25,000 units at snack time.

In order to reduce this burden and increase patient compliance, it is desirable to provide higher activities of lipases.

Another problem is that, in general, commercially available lipase or PERT products are not specifically adapted to the environmental conditions of the human small intestine, including pH and bile acid concentration and composition. The latter 2 parameters may vary significantly between humans and e.g. pigs.

Background

It is therefore an object of the present invention to provide better treatment options for patients with lipid digestion deficiencies or disorders such as pancreatic insufficiency (EPI).

It is another object of the present invention to provide an alternative to conventional Pancreatic Enzyme Replacement Therapy (PERT).

These and other objects are met by the methods and means according to the independent claims of the present invention. Dependent claims relate to specific embodiments.

Summary of The Invention

The present invention provides modified lipases. The general advantages of the invention and its features are discussed in detail below.

Brief description of the drawings

FIG. 1: the activities of Wild Type (WT) and Q55K mutant were compared together with 3mm 86339 taurocholate.

FIG. 2 is a schematic diagram: the activity of WT and Q55K mutants was compared with 7.5mm EPS0900000 taurocholate.

FIG. 3: the specific lipase activity of the purified WT-and Q55K mutated lipase was altered by increasing the mixed bile salt concentration and 140mM NaCl.

FIG. 4: bile salt concentration in the human small intestine. Taken from Northfield et al, mcColl,1973. In all fractions, except the lower ileum, the total bile acid concentration was 4. Mu.M/ml or more (equal to 4mM or more).

FIG. 5 is a schematic view of: pH profile of human small intestine. Taken from Koziolek et al, 2015.SBTT _norm = normalized Small Bowel Transit Time (SBTT) between gastric emptying time and colon arrival time. This also means that the lipase of the present invention shows higher activity compared to WT lipase under conditions reflecting in vivo conditions.

FIG. 6: a vector map of the pMA vector used, showing the target gene (Tt 00320120), the origin of replication (Ori), ampicillin resistance (AmpR) and various restriction sites (PsiI et al). pMA was generated from the pMX system.

FIG. 7: a vector diagram of the pAX vector used shows the expression cassettes (promoter: MTT1, target gene: ttherm-00320120, terminator: BTU 2), 3 resistances: chloramphenicol (CmR), ampicillin (AmpR), codon-harmonized paromomycin resistance sequence (ha _ NeoR), 2 Cre/loxP sites (loxP), 3 origins of replication (colE 1: E.coli, rDNA ori: tetrahymena thermophila (T.thermophila)) and different restriction sites (EcoRV et al). The backbone is the pUC119 vector.

Detailed Description

Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular components of the devices or process steps of the methods described, as such devices and methods may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be understood that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include singular and/or plural referents unless the context clearly dictates otherwise. Further, it is to be understood that where ranges of given parameters are separated by numerical values, the ranges are to be considered as inclusive of the recited limits.

It should also be understood that the embodiments disclosed herein are not meant to be construed as separate embodiments not relating to each other. Features discussed with one embodiment are meant to be disclosed in relation to other embodiments also shown herein. If, in one instance, a particular feature is not disclosed with one embodiment but with another, the skilled artisan will understand that it is not necessary to imply that the feature is not intended to be disclosed with the other embodiment. The skilled artisan will appreciate that the gist of the present application is in disclosing the features, as well as other embodiments, which have not yet been done merely for the purpose of illustration and to maintain the specification at a manageable space.

Further, the contents of the prior art documents referred to herein are incorporated by reference. Especially with respect to prior art documents, which disclose standard or conventional methods. In this case, the main purpose of incorporation by reference is to provide sufficient enabling (enabling) disclosure and to avoid lengthy repetition.

According to a first aspect of the invention there is provided a lipase comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO 1, with the proviso that the lipase has a substitution at position Q55 of an amino acid with a basic side chain at neutral pH.

It is understood that in the lipase sequence according to SEQ ID NO 1, the signal peptide (= leader peptide) is absent. One suitable signal peptide is disclosed as SEQ ID NO 9, having a length of 6 amino acid residues.

All amino acid positions mentioned in this application relate to the numbering in SEQ ID NO 1. However, in case the lipase has an N-terminal signal peptide (= leader peptide) as in SEQ ID NO 9, the counting needs to take into account the extra N-terminal amino acid residue of the signal peptide.

The term "amino acid having a basic side chain at neutral pH" as used herein encompasses, for example, amino acids having an NH group in the side chain, which NH group carries a positive charge at neutral pH.

In one embodiment, the substitution is by an amino acid selected from the group consisting of lysine (K), arginine (R), and histidine (H).

Alternatively, the lipase comprises SEQ ID NO 1 or a functional fragment of a sequence having at least 90% sequence identity to SEQ ID NO 1, which fragment encompasses position Q55 and has said substitution.

Such fragments may, for example, have AA residues 21-256 of SEQ ID NO 1 or a sequence having at least 90% sequence identity to SEQ ID NO 1, including substitution of Q55.

The term "functional" means that such fragments retain lipase activity, particularly under the pH-and bile salt conditions set forth herein.

The fragment preferably has a minimum length of 100 or more amino acid residues (AA); more preferably 110AA or more; not less than 120AA; not less than 130AA; more than or equal to 140AA; not less than 150AA; more than or equal to 160AA; 170AA or more; more than or equal to 180AA; more than or equal to 190AA; more than or equal to 200AA; more than or equal to 210AA; more than or equal to 220AA; 230AA or more; more than or equal to 240AA; more than or equal to 250AA; more than or equal to 260AA; more than or equal to 270AA; and most preferably 280AA or more.

The fragment preferably can have a maximum length of 280AA or less; more preferably 270AA or less; less than or equal to 260AA; less than or equal to 250AA; less than or equal to 240AA; less than or equal to 230AA; less than or equal to 220AA; less than or equal to 210AA; less than or equal to 200AA; less than or equal to 190AA; less than or equal to 180AA; less than or equal to 170AA; less than or equal to 160AA; less than or equal to 150AA; less than or equal to 140AA; less than or equal to 130AA; less than or equal to 120AA; less than or equal to 110AA; and most preferably ≦ 100AA.

SEQ ID NO 1 is the amino acid sequence of the ciliate Tetrahymena thermophila, designated TTHERM _00320120 (UniProtKB-Q237S 4 (Q237S 4_ TETTS). In Brock et al, 2016, the use thereof in the treatment of pancreatic insufficiency is discussed.

Surprisingly, the inventors have shown that the claimed lipase has an increased lipolytic activity in a medium with a pH of > 5.5, in a medium with a bile salt concentration of > 2.5mM, and/or in a medium containing a mixture of 2 or more different bile acids compared to a lipase having the amino acid sequence of SEQ ID NO 1.

These conditions reflect in vivo conditions in the human small intestine.

The terms bile salt and bile acid are used interchangeably herein. Bile acids are steroid acids found primarily in mammalian bile. In humans, taurocholic and glycocholic acids (derivatives of cholic acid) and taurochenodeoxycholic and glycochenodeoxycholic acids (derivatives of chenodeoxycholic acid) are the major bile salts in bile and at approximately equal concentrations. It has also been found that 7-alpha-dehydroxy derivatives thereof, conjugated salts of deoxycholic and lithocholic acids, and derivatives of cholic, chenodeoxycholic and deoxycholic acids, account for more than 90% of human bile acids. Bile acids form about 80% of the organic compounds in bile. The primary function of bile acids is to allow digestion of dietary fats and oils as a surfactant that emulsifies them into micelles (hydrophobic side facing fat and hydrophilic side facing outward). The hydrophilic side is negatively charged, which prevents the bile-coated fat droplets from reaggregating into larger fat particles. Micelles in the duodenum often have a diameter of about 14-33 μm.

The dispersion of food fat into micelles provides a greatly increased surface area for pancreatic lipase activity, which actually digests triglycerides and can reach the fat core through gaps between bile acids.

In some embodiments, the lipase of the invention has sequence identity of ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98% or most preferably ≥ 99% with SEQ ID NO 1, provided that the lipase is implemented with a substitution at position Q55.

In some embodiments, the lipase of the invention has sequence identity of ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98 or most preferably ≥ 99% with either SEQ ID NO 3 ("Q55K"), SEQ ID NO 4 ("Q55R") or SEQ ID NO 5 ("Q55H").

In some embodiments, the lipase of the invention is identical to SEQ ID NO 1 except that the lipase has a substitution at position Q55. Thus, in some embodiments, the lipase is identical to SEQ ID NO 1 except that the lipase has the substitution Q55K (SEQ ID NO 3). In some embodiments, the lipase is identical to SEQ ID NO 1 except that the lipase has the substitution Q55R (SEQ ID NO 4). In some embodiments, the lipase is identical to SEQ ID NO 1 except that the lipase has the substitution Q55H (SEQ ID NO 5).

The substitution at position Q55 replaces glutamine (Q) with lysine (K), arginine (R), or histidine (H), all of which carry a net positive charge, which are aliphatic, uncharged amino acids.

In general, many lipases have a catalytic triad in their active center, which contains aspartic acid, histidine and serine. Aspartic acid extracts a proton from histidine and activates it. In response, the catalytically active histidine recruits a proton from serine, which in turn increases the nucleophilicity of the serine residue. The latter can now attack the carbonyl carbon of the substrate ester located at the active center, which forms part of the enzyme's fatty substrate.

The inventors show that the serine residue in position Q55 from the catalytic triad is only

Without being bound by theory, substitution of neutral Q55 with a positively charged amino acid residue may actually be responsible for the observed increase in activity.

In one embodiment, the lipase of the invention retains a catalytic triad comprising aspartate (D), histidine (H) and serine (S) after having at least 90% sequence identity to SEQ ID NO 1 or a fragment thereof as described elsewhere herein, and having a Q55 substitution. In one embodiment, the catalytic triad comprises the following amino acid residues of SEQ ID NO 1: s140, D199, and H256.

In one embodiment, the lipase of the invention retains the oxygen ion pocket that stabilizes the intermediate product after having at least 90% sequence identity to SEQ ID NO 1 or being a fragment thereof as described elsewhere herein, and having a Q55 substitution, optionally after retaining the catalytic triad as described elsewhere herein. The pocket is mainly formed by the following amino acid residues according to SEQ ID NO 1 Tyr 21 and Thr76 (optionally plus S140 and H256).

Other lipases having at least 90% sequence identity with SEQ ID NO 1 are those according to SEQ ID Nos 10-12 (with Q55K mutation), SEQ ID Nos 13-15 (with Q55R mutation) and SEQ ID Nos 16-18 (with Q55H mutation).

"percent sequence identity" is determined by comparing 2 optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence within the comparison window can include additions or deletions (i.e., gaps) as compared to a reference sequence (e.g., a polypeptide) that does not comprise additions or deletions, for optimal alignment of the 2 sequences. The percentages are calculated as follows: the number of positions at which the identical nucleic acid base or amino acid residue occurs in 2 sequences is determined to yield the number of matched positions, which is divided by the total number of positions in the window of comparison and the result multiplied by 100 to yield the percentage of sequence identity.

The term "identical" or percent "identity" in the context of 2 or more nucleic acid or polypeptide sequences relates to 2 or more sequences or subsequences that are the same in sequence. When compared and aligned for maximum correspondence in a comparison window or designated region, 2 sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are identical (i.e., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity in the designated region, or are not specified, in the complete sequence of the reference sequence), as determined by one of the following sequence comparison algorithms or by manual alignment and visual inspection.

One suitable algorithm for determining sequence identity is the BLAST algorithm. Another algorithm for determining sequence identity is the Clustal Omega algorithm.

The present disclosure provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides exemplified herein, respectively. Optionally, identity exists over a region of at least about 15, 25 or 50 nucleotides in length, or more preferably over a region of 100-500 or 1000 or more nucleotides in length, or over the full-length reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region of at least 5, 10, 15, or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75, or 100 amino acids in length, optionally at least about 150, 200, or 250 amino acids in length, or in a full-length reference sequence. With respect to shorter amino acid sequences, such as amino acid sequences of 20 or fewer amino acids, substantial identity exists when 1 or 2 amino acid residues are conservatively substituted, as defined herein.

According to one embodiment, the lipase has the substitution Q55K.

According to another embodiment, the lipase has a lipolytic activity of at least 30,000u/g.

In the context of the present disclosure, the terms "lipase activity" and "lipolytic activity" are used interchangeably. To determine lipase activity (lipolytic activity), a modified version of the colorimetric assay described elsewhere herein from Nixon and Chan (1979) can be used.

According to another embodiment, the lipase comprises at least one conservative amino acid substitution in addition to the substitution at position Q55, compared to the amino acid according to SEQ ID NO 1.

In this context, a "conservative amino acid substitution" has less effect on lipase function than a non-conservative amino acid substitution. Although there are many ways to classify amino acids, amino acids are generally divided into 6 main groups based on their structure and the general chemical characteristics of their R groups.

In some embodiments, a "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains are defined in the art. These families include amino acids with:

basic side chains (e.g.lysine, arginine, histidine),

acidic side chains (e.g.aspartic acid, glutamic acid),

uncharged polar side chains (e.g.glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),

nonpolar side chains (e.g.alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),

beta branched side chains (e.g. threonine, valine, isoleucine) and

aromatic side chains (e.g.tyrosine, phenylalanine, tryptophan, histidine).

Other conservative amino acid substitutions can also occur in amino acid side chain families, such as when asparagine is substituted for aspartic acid to modify the charge of the peptide. Conservative variations can also include chemically homologous unnatural amino acids (i.e., synthetic unnatural hydrophobic amino acids substituted for leucine, synthetic unnatural aromatic amino acids substituted for tryptophan).

SEQ ID Nos 6-8 show the wild type sequence variants with 2 conservative amino acid substitutions, i.e., V70I/V152I, respectively; V71I/L207I or V119I/Y168F. Mutations at position Q55 as listed herein can also be achieved in these wild type variants.

According to another embodiment, the lipase has an increased lipolytic activity in a medium at pH.gtoreq.5 compared to a lipase having the amino acid sequence of SEQ ID NO 1.

In one embodiment, such increased lipolytic activity is present at a pH between ≥ 5.5 and ≤ 11, between ≥ 6 and ≤ 10 or between ≥ 6.5 and ≤ 9, as compared to the lipase having the amino acid sequence of SEQ ID NO 1.

According to another embodiment, the lipase has an increased lipolytic activity in a medium with a total bile salt concentration ≥ 2.5mM, compared to the lipase having the amino acid sequence SEQ ID NO 1.

In one embodiment, the increased lipolytic activity is present in a medium having a total bile salt concentration of between ≥ 2.5mM to ≤ 15mM, as compared to the lipase having the amino acid sequence of SEQ ID NO 1.

In various embodiments, the bile salt is selected from the group consisting of cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, and lithocholic acid.

According to another embodiment, the lipase has increased lipolytic activity in a medium comprising a mixture of 2 or more different bile salts compared to a lipase having the amino acid sequence of SEQ ID NO 1.

In various embodiments, the 2 or more different bile salts are selected from cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, and lithocholic acid.

In any of the 3 cases, the active energy is increased by more than or equal to 5%,. Gtoreq.10%,. Gtoreq.15%,. Gtoreq.20%,. Gtoreq.25%,. Gtoreq.30%,. Gtoreq.40%,. Gtoreq.50%,. Gtoreq.60%,. Gtoreq.70%,. Gtoreq.80%,. Gtoreq.90%,. Gtoreq.100%,. Gtoreq.150% or even more than or equal to 200%. To determine lipase activity (lipolytic activity), a modified version of the colorimetric assay described elsewhere herein from Nixon and Chan (1979) can be used.

According to a further aspect of the invention there is provided a nucleic acid encoding a lipase according to any of the above embodiments. Such nucleic acids may be, for example, mRNA or cDNA. Suitable vectors comprising such nucleic acids are also provided.

According to another embodiment, there is provided the use of the lipase described above for the treatment of a human or animal subject (for the manufacture of a medicament for said treatment), said subject

The diagnosis is that,

is suffering from or has

At risk of developing

A deficiency in lipid digestion, a digestive disorder, and/or an inflammatory disorder, or for the prevention of such a disorder.

According to one embodiment, the digestive disorder is exocrine pancreatic insufficiency. Such pancreatic exocrine insufficiency may result, inter alia, from cystic fibrosis, pancreatic duct obstruction, or pancreatectomy.

According to another embodiment, the inflammatory disorder is chronic inflammation of the pancreas (pancreatitis) or inflammatory bowel disease.

According to another aspect of the invention, there is provided a pharmaceutical composition comprising a lipase as described above and optionally one or more pharmaceutically acceptable excipients.

According to another aspect of the present invention there is provided a combination comprising (i) a lipase or a pharmaceutical composition as described above and (ii) one or more therapeutically active compounds.

According to another aspect of the present invention there is provided a method of treating or preventing a lipid digestion deficiency, a digestion disorder and/or an inflammatory condition, the method comprising administering to a human or animal subject a therapeutically sufficient dose of (i) a lipase, (ii) a pharmaceutical composition or (iii) a combination as described above.

According to another aspect of the invention, a therapeutic kit of parts is provided comprising:

a) (ii) the lipase as described above, (ii) a pharmaceutical composition or (iii) a combination,

b) A device for applying the composition, composition or combination, and

c) Instructions for use.

Such devices are, for example, capsules, pills, syringes, inhalers, etc.

According to another aspect of the present invention, there is provided a method for producing lipase, the method comprising the steps of

a) Expressing said lipase in an expression host from the order ciliates, and

b) Purifying the lipase expressed in step a).

According to one embodiment, the method comprises the step of transforming ciliates with a vector encoding the lipase prior to step a). Suitable carriers are disclosed elsewhere herein.

Methods for transforming ciliates can be used in the context of the present invention, including microinjection, electroporation, and particle bombardment, and are described, for example, in Tondravi & Yao (1986), gaertig & Gorovsky (1992), and Cassidy-Hanley et al (1997).

Methods of transformation and heterologous protein expression are described for some protists (WO 00/58483 and WO 00/46381). Mitotically stable transformants of the ciliate tetrahymena thermophila can be generated after bombardment of the large or small germ nuclei of the transformants by microinjection, electroporation or particles.

Selection of transformants can be performed with different selectable markers such as neomycin resistance (Weide et al, 2006, BMC) and integration of heterologous genes by homologous DNA recombination, which results in stable thymidine auxotrophic Tetrahymena cells (Weide et al, 2006, BMC). In addition, resistance to blasticidin S (Weide et al, 2007, BMC) or paclitaxel (WO 00/46381) is also contemplated.

Suitable promoters for lipase expression in ciliates are disclosed for example in US2008261290A1, which is the applicant's registration of the present invention, the content of which is incorporated herein by reference. Among these, heat-inducible promoters and metallothionein promoters are disclosed, which can also be used for the purpose of the present invention.

According to another embodiment, the expression host is from the genus tetrahymena.

According to another embodiment, the expression host is tetrahymena thermophila.

Examples

The invention is illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative or exemplary and not restrictive in character; the present invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims shall not be construed as limiting the scope.

All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown at 5'- > 3'.

Materials and methods

Culture medium:

LB (Lysogeny Broth) medium

10g/L casein peptone

5g/L Yeast extract

5g/L g/L NaCl

Dissolving in distilled water, pH 7.5

LB agar plate

15g/L agar-agar

Dissolved in LB culture medium

Optional 25. Mu.g/mL kanamycin or 100. Mu.g/mL ampicillin

Cre-Lox-LB agar plate

7% (w/v) sucrose

30 ug/mL chloramphenicol

100 ug/mL ampicillin

86% (v/v) LB with 15g/L agar

Super Optimal Broth (SOC) medium, invitrogen GmbH, carlsreue, germany

Dryls(1x)

1.5mM trisodium citrate dihydrate

1mM NaH ₂ PO ₄ -monohydrate

1mM Na ₂ HPO ₄

1.5mM CaCl ₂

MW 1010

10g/L malt extract

10g/L wheat peptone E1

5g/L Yeast extract

2g/L glucose monohydrate

1mL/L ferrous sulfate/chelate solution

MW 1515

15g/L malt extract

15g/L wheat peptone E1

5g/L Yeast extract

2g/L glucose monohydrate

1mL/L ferrous sulfate/chelate solution

Concentration of paromomycin used for fermentation:

224 mug/ml paromomycin

Paromomycin concentrations for mutant adaptation:

196 mug/ml paromomycin.

Buffer solution

As buffers, typical buffers for hydrophobic interaction chromatography are used, as disclosed for example in Djogo et al 1999, the content of which is incorporated herein by reference.

Machine and column

1. Mutation of expression vectors

The MTT1_ TTHERM _00320120 gene (UniProtKB-Q237S 4, the amino acid of which is shown herein as SEQ ID NO 1) in pAM vector was used for mutagenesis, which was based on the QuikChange method (QUIKCHANGE) ^TM Directed mutagenesis kit from Stratagene, cat 200518, loke et al 2001). 2 primers with point mutations were generated to generate amino acid substitutions. Following gene mutation in the pMA vector, the genes were ligated in the pDL _ S2 vector and then inserted into the shuttle vector pAX _ ha _ neo using the Cre-dependent recombinase system. See figures 6 and 7 for each vector.

2. Culture of Tetrahymena and expression plasmid transformation (biolistic bombardment)

For this experiment, tetrahymena thermophila strains 1868/4 and 1868/7 were used for transformation, which was carried out as described previously by Cassidy-Hanley et al (1997). The cultivation was carried out in a 1l bioreactor for 50 hours in MW1515 medium (15 g/l malt extract, 15g/l wheat peptone E1,5g/l yeast extract, 1ml/l ferrous sulfate/chelate solution, 2g/l glucose monohydrate). In mid-log phase, cdCl2 was added at a final concentration of 10. Mu.g/ml to induce expression through the MTT1 promoter, approximately 24 hours after inoculation. Correct expression was tested by coomassie stained SDS-PAGE and WB using polyclonal antibody.

3. Purification of

To determine the specific activity in some measurements, the lipase was purified stepwise through multiple steps. The supernatant was concentrated by dialysis and the buffer was exchanged. After 20x concentration, the sample was diluted with phosphate buffer for binding on a Hydrophobic Interaction Chromatography (HIC) column. After washing the column, one-step elution was performed by gradually decreasing the ammonium sulfate concentration to 0%.

The eluted protein was collected and used for further purification on a Superdex75 Increatase 10/300 GL Size Exclusion Chromatography (SEC) column.

4. Determination of Lipase Activity

To determine lipase activity (lipolytic activity), a modified version of the colorimetric assay from Nixon and Chan (1979) was used as described by Brock et al 2016, the contents of which are incorporated herein by reference. For the experiments, different bile acids and defined mixtures of bile salts were tested: taurocholate (3 mM) was used for standards as well as samples, 7.5mM taurocholate (BRP) and mixed bile salt solution (MBS) were used only for samples as described by Gargouri et al (1986). The pH was adjusted by 54mM and pH 6-7.5 phosphate buffer, which contained 140mM NaCl to final concentration.

Example 1:

FIG. 1: the activity of Wild Type (WT) and Q55K mutant were compared together with 3mM taurocholate.

For initial use of 97% pure Sigma (Sigma) 3mM taurocholate 86339, quantification of normalized volume by WB, WT-0120 lipase activity differed from the Q55K mutant lipase only in that the activity was higher from pH 4 to pH 8. Taurocholate concentrations did not reach Critical Micelle Concentration (CMC) and did not start to inhibit the activity at alkaline pH.

FIG. 2: the activity of WT and Q55K mutants was compared with 7.5mm EPS0900000 taurocholate.

Other taurocholates are preferably from european pharmacopoeia, EPS0900000 BRP (from LGC), have no existing purity evidence and are brown in color, so that there is a completely different distribution of activity over pH. Using a concentration of 7.5mM, 2 lipases, WT and Q55K lipase, showed very similar activity up to pH 6, but then WT activity sagged, while Q55K activity was able to maintain pH 6 levels and remain above WT at pH 7 and pH 8.

Without being bound by theory, this effect appears to be based on impurities in the second taurocholate salt, which just means that other bile acids are present, the CMC being much lower than that of taurocholate. These bile acids apparently were able to inhibit WT lipase activity at alkaline pH.

In fact, the medium containing the bile acid mixture reflects the in vivo situation much better than the medium containing only pure taurocholate.

FIG. 3: the specific lipase activity of the purified WT-and Q55K mutant lipases was altered by increasing the mixed bile salt concentration and 140mM NaCl.

Analysis of purified WT and Q55K lipase at different MBS concentrations closer to in vivo conditions showed strong effect at pH 7. The specific activity of the 2 enzymes is first increased, correlated with MBS concentrations, where the WT-lipase starting activity is higher. The activity of the Q55K lipase exceeded the WT-activity only after reaching MBS concentrations between 2.5-5.0mM, demonstrating that the Q55K mutation is more resistant to high bile salt concentrations at neutral to alkaline pH.

FIG. 4 is a schematic view of: bile salt concentration in the human small intestine. Taken from Northfield et al, mcColl,1973. In all fractions, except the lower ileum, the total bile salt concentration was ≥ 4. Mu.M/ml (equivalent ≥ 4 mM)

This means that the lipase of the present invention shows higher activity under conditions reflecting in vivo conditions as compared to WT lipase.

FIG. 5 is a schematic view of: pH profile of human small intestine. Taken from Koziolek et al, 2015.SBTT _norm = normalized Small Bowel Transit Time (SBTT) between gastric emptying time and colon arrival time. This also means that the lipase of the present invention shows higher activity compared to WT lipase under conditions reflecting in vivo conditions.

Reference documents

Cassidy-Hanley,D.et al.(1997)Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment,Genetics,146(1),pp.135–147

Gargouri,Y.et al.(1986)‘Human gastric lipase.The effect of amphiphiles.’,Eur J Biochem,156(2),pp.305–310

Nixon,M.and Chan,S.H.(1979)‘A simple and sensitive colorimetric method for the determination of long-chain free fatty acids in subcellular organelles.’,Anal Biochem,97(2),pp.403–409.

QuickChange Protocol:

http://kirschner.med.harvard.edu/files/protocols/Stratagene_quickchangepdf.pdf

Loke P,Sim TS.A comparison of three site-directed mutagenesis kits.Z Naturforsch C.2001

Sep-Oct；56(9-10):810-3

Northfield TC,McColl I.Postprandial concentrations of free and conjugated bile acids down the length of the normal human small intestine.Gut.1973Jul；14(7):513-8

Koziolek M et al,Investigation of pH and Temperature Profiles in the GI Tract of Fasted Human Subjects Using the Intellicap System.J Pharm Sci.2015Sep；104(9):2855-63

Brock et al.,Novel ciliate lipases for enzyme replacement during exocrine pancreatic insufficiency.Eur J Gastroenterol Hepatol.2016Nov；28(11):1305-12

Tondravi MM,Yao MC.Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes.PNAS 1986Jun；83(12):4369-73

Gaertig J,Gorovsky MA.Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants.PNAS 1992Oct 1；89(19):9196-200

Diogo MM,Silva S,Cabral JM,Queiroz JA.Hydrophobic interaction chromatography of Chromobacterium viscosum lipase J Chromatogr A.1999Jul 23；849(2):413-9.

Weide et al.,A recombinase system facilitates cloning of expression cassettes in the ciliate Tetrahymena thermophila.BMC Microbiology 2007,7:12

Weide et al.,Secretion of functional human enzymes by Tetrahymena thermophila.BMC Biotechnol.2006；6:19.

WO2007006812A1

WO 00/58483

WO 00/46381

WO 00/46381

Sequence of

The following sequences form part of the disclosure of this application. The present application also provides a WIPO ST 25 compatible electronic sequence listing. For the avoidance of doubt, if there are differences between the sequences in the table below and the electronic sequence list, the sequences in this table should be considered correct.

Sequence listing

<110> Kyolaan shares Co

<120> novel lipase

<130> CD 42798

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 272

<212> PRT

<213> Tetrahymena thermophila

<400> 1

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Gln Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 2

<211> 867

<212> DNA

<213> Tetrahymena thermophila

<400> 2

atgaaattgt aattgcttct attggtttgc ttgtcatttg ctgcctgcta atcatttact 60

tatacttaat cacttgctta agacttagct ggtttctctc ttgcttctta ctgtaatcct 120

aaatctatag aacaatggaa ttgtggatgt gcttgtgata aaaaccctta aggacttcga 180

aatgttacta tcttatttaa ctctactcta taagctagtg gatatttagg ctactccact 240

catcatgatg caattgttgt tgtattcaga ggaacagtac cttggttaat cgaaaattgg 300

attgctgact taaacacctt caagacttag tacccactct gccaaaactg ttatgtccat 360

taaggctttt ataaccagtt caaataattg aaatctcagc ttgttactag ctttacttca 420

cttcgttaac tatatcctaa tgcaaaagta tttgttacag gacattctct tggtgctgca 480

atgagtgctc actcaatacc agtaatttac taattaaatg gaaataaacc tattgatgct 540

ttttacaatt atggttgtcc tagagtaggt gactaaactt atgcaaactg gtttaacagt 600

taaaattttg ccttagaata tggtagaatt aataatgctg ctgatccagt tcctcattta 660

cctcctcttc tttacccatt ttcatttttc cactacaacc atgaaatatt ctatccttct 720

tttgttcttt ttggaaacta acataactaa tgttaaaacg cggaaacaat atttggtgca 780

gatggagtaa taatagcagc taatgttcta gaccatctaa cttattttgg atgggattgg 840

tctggttcta tattaacttg ctaatga 867

<210> 3

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 3

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Lys Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 4

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 4

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Arg Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 5

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 5

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu His Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 6

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 6

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Gln Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Ile Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Ile Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 7

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 7

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Gln Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Ile Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Ile Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 8

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 8

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Gln Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Ile Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Phe Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 9

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> Lead peptide

<400> 9

Met Lys Leu Gln Leu Leu Leu Leu Val Cys Leu Ser Phe Ala Ala Cys

1 5 10 15

<210> 10

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 10

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Lys Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Ile Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Ile Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 11

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 11

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Lys Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Ile Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Ile Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 12

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 12

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Lys Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Ile Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Phe Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 13

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 13

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Arg Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Ile Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Ile Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 14

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 14

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Arg Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Ile Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Ile Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 15

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 15

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu Arg Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Ile Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Phe Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 16

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 16

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu His Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Ile Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Ile Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 17

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 17

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu His Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Ile Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Val Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Ile Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

<210> 18

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> Lipase mutant

<400> 18

Gln Ser Phe Thr Tyr Thr Gln Ser Leu Ala Gln Asp Leu Ala Gly Phe

1 5 10 15

Ser Leu Ala Ser Tyr Cys Asn Pro Lys Ser Ile Glu Gln Trp Asn Cys

20 25 30

Gly Cys Ala Cys Asp Lys Asn Pro Gln Gly Leu Arg Asn Val Thr Ile

35 40 45

Leu Phe Asn Ser Thr Leu His Ala Ser Gly Tyr Leu Gly Tyr Ser Thr

50 55 60

His His Asp Ala Ile Val Val Val Phe Arg Gly Thr Val Pro Trp Leu

65 70 75 80

Ile Glu Asn Trp Ile Ala Asp Leu Asn Thr Phe Lys Thr Gln Tyr Pro

85 90 95

Leu Cys Gln Asn Cys Tyr Val His Gln Gly Phe Tyr Asn Gln Phe Lys

100 105 110

Gln Leu Lys Ser Gln Leu Ile Thr Ser Phe Thr Ser Leu Arg Gln Leu

115 120 125

Tyr Pro Asn Ala Lys Val Phe Val Thr Gly His Ser Leu Gly Ala Ala

130 135 140

Met Ser Ala His Ser Ile Pro Val Ile Tyr Gln Leu Asn Gly Asn Lys

145 150 155 160

Pro Ile Asp Ala Phe Tyr Asn Phe Gly Cys Pro Arg Val Gly Asp Gln

165 170 175

Thr Tyr Ala Asn Trp Phe Asn Ser Gln Asn Phe Ala Leu Glu Tyr Gly

180 185 190

Arg Ile Asn Asn Ala Ala Asp Pro Val Pro His Leu Pro Pro Leu Leu

195 200 205

Tyr Pro Phe Ser Phe Phe His Tyr Asn His Glu Ile Phe Tyr Pro Ser

210 215 220

Phe Val Leu Phe Gly Asn Gln His Asn Gln Cys Gln Asn Ala Glu Thr

225 230 235 240

Ile Phe Gly Ala Asp Gly Val Ile Ile Ala Ala Asn Val Leu Asp His

245 250 255

Leu Thr Tyr Phe Gly Trp Asp Trp Ser Gly Ser Ile Leu Thr Cys Gln

260 265 270

Claims (19)

1. A lipase comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO 1, or comprising a functional fragment thereof encompassing position Q55,

provided that the lipase has a substitution at position Q55 with an amino acid with a basic side chain at neutral pH.

2. The lipase of claim, wherein such substitution is by an amino acid selected from lysine (K), arginine (R) and histidine (H).

3. The lipase of claim 1 and/or 2, having the substitution Q55K.

4. The lipase of any preceding claim, having lipolytic activity of at least 30,000u/g.

5. The lipase of any one of the preceding claims, comprising at least one conservative amino acid substitution other than the substitution at position Q55, compared to the amino acid according to SEQ ID NO 1.

6. The lipase according to any of the preceding claims, which has an increased lipolytic activity in a medium at a pH ≥ 5.5, as compared to the lipase having the amino acid sequence SEQ ID NO 1.

7. The lipase according to any of the preceding claims, which has an increased lipolytic activity in a medium with a total bile salt concentration of > 2.5mM compared to the lipase having the amino acid sequence of SEQ ID NO 1.

8. The lipase according to any of the preceding claims, which has an increased lipolytic activity in a medium comprising a mixture of 2 or more different bile acids compared to the lipase having the amino acid sequence of SEQ ID NO 1.

9. A nucleic acid encoding the lipase of any preceding claim.

10. Use of a lipase as defined in any of claims 1-8 for the treatment of a human or animal subject (for the manufacture of a medicament for said treatment), said subject

The result of the diagnosis is,

is suffering from or is

At risk of developing

A lipid digestion deficiency, a digestion disorder, or an inflammatory disorder, or for preventing such a disorder.

11. The use according to claim 10, in which the digestive disorder is exocrine pancreatic insufficiency.

12. A pharmaceutical composition comprising the lipase of any one of claims 1-8 and optionally one or more pharmaceutically acceptable excipients.

13. A combination comprising (i) the lipase of any one of claims 1-8 or the pharmaceutical composition of claim 12 and (ii) one or more therapeutically active compounds.

14. A method of treating or preventing a lipid digestion deficiency, a digestion disorder or an inflammatory disorder, the method comprising administering to a human or animal subject a therapeutically sufficient dose of (i) the lipase of any one of claims 1-8, (ii) the pharmaceutical composition of claim 12 or (iii) the combination of claim 13.

15. A therapeutic kit-of-parts comprising:

a) (i) the lipase of any one of claims 1-8, (ii) the pharmaceutical composition of claim 12 or (iii) the combination of claim 13,

b) A device for applying said composition, composition or combination, and

c) Instructions for use.

16. A method for producing the lipase of any one of claims 1-8, comprising the steps of

a) Expressing the lipase in an expression host from the order ciliates, and

b) Purifying the lipase expressed in step a).

17. A method according to claim 16, comprising the step of transforming ciliates with a vector encoding the lipase prior to step a).

18. The method of any one of claims 16 and/or 17, wherein the expression host is from the genus tetrahymena.

19. The method of any one of claims 16-18, wherein the expression host is Tetrahymena thermophila.

Images